This invention relates to the discovery of nourseothricin (NST) drug sensitivity in the pathogenic yeast, Candida albicans and in Saccharomyces cerevisiae. In particular, the present invention relates to a cognate drug resistance marker system for use in gene transformation and disruption experimentation. Specifically, the present invention provides a modified nourseothricin/streptothricin resistance gene, SAT, for expression in C. albicans. The present invention also provides a cell, nucleic acid molecule, and vector comprising the modified SAT1 nucleic acid sequence. The present invention further provides a SAT expression module for gene knock-outs.
Opportunistic fungi are a rapidly emerging class of microbial pathogens causing systemic fungal infection or xe2x80x9cmycosisxe2x80x9d in patients immunocompromised either by illness (e.g., AIDS) or standard medical treatment (e.g., organ transplants, chemotherapy, radiation therapy). Candida spp. rank as the predominant genus of such fungal pathogens. In recent years, rapid and reliable diagnosis of fungal infection has advanced primarily through the application of molecular biological techniques. Understanding the pathogenesis of this organism, from which novel treatment strategies will develop, is also dependent on improved techniques in molecular genetics.
The recent commitment by the Stanford Sequence Center to sequence the entire C. albicans yeast genome will accelerate our understanding in both the biology and eventual treatment of candidiasis. The DNA sequence resulting from this enterprise however offers only a prediction towards potential pathogenesis pathway(s) and antifungal targets. Maximum information gained from this effort requires experimentation. The ability to study the role of any particular gene, both by abolishing its function through gene disruption experiments, as well as overproducing its gene product through transformation experiments, directly tests the predictions made by bioinformatic analysis. As C. albicans is an imperfect fungus which lacks a sexual cycle and is fixed in the diploid state, gene disruption experiments are more cumbersome, requiring replacement of both alleles of the target gene before an examination of its null phenotype be determined. To this end, improved DNA methodologies are required for experimentation in C. albicans. 
Currently, auxotrophic markers are employed to select for precise genetic alterations in C. albicans. Auxotrophic markers are recessive mutations, usually in biosynthetic genes, which can be complemented by either supplementing the yeast strain with the desired requirement (e.g., uridine) or by transformation of the wild type gene. A number of non-reverting, auxotrophic mutations, to which the complementing wild type gene has been cloned, are available for genetic manipulations in C. albicans (Pla et al., 1996 Yeast 12:1677-1702). CAI4, the standard C. albicans strain employed by researchers, contains a single auxotrophic markerxe2x80x94a homozygous null mutation in the CaURA3 gene. The utility of this strain stems largely from a xe2x80x9cURA-blasterxe2x80x9d gene disruption procedure developed for C. albicans by Fonzi and Irwin (1993 Genetics 134:717-728) which utilizes a CaURA3 gene flanked by direct repeats of the Salmonella typhimurium HisG gene. This Ura-blaster cassette is used to replace part of the target gene in vitro. The resulting disruption cassette is then transformed into CAI4, whereby through homologous recombination, Ura+ transformants harboring a heterozygous mutation for the target gene are selected. Counterselection on 5-fluoroorotic acid (5-FOA), relying on intrachromosomal recombination between HisG repeats, excises the CaURA3 gene, leaving a single copy of the HisG sequence within the target gene, and allowing reuse of the auxotrophic marker-based disruption cassette for disruption of the target gene""s second allele.
Despite a reliance on auxotrophic markers to select for successful DNA transformation or gene disruption, this dependency comes with significant limitations. Firstly, analysis is restricted to the genetic background to which the auxotrophic mutation has been introduced and the complementing gene available. This severely restricts genetic analyses of clinical isolates which lack auxotrophic markers. Alternatively, a specially constructed strain containing the appropriate auxotrophic marker must first be constructed, a procedure which is both time consuming and problematic. A second common problem associated with auxotrophic markers is the limited number of stable mutations constructed in a particular strain background. As outlined above, CAI4, the most widespread C. albicans strain used for genetic manipulation, maintains only a single auxotophic marker. Although, the URA3 marker can be reused in gene disruption experiments, this process has significant drawbacks, and more sophisticated manipulations (for example, the selection and stable maintenance of a second gene) are difficult. Auxotrophic mutations also potentially affect physiological processes such as pathogenicity, rendering the strain inappropriate for virulence studies (Pla et al., 1996, Yeast 12:1677-1702). Therefore, a strain maintaining multiple auxotrophic mutations must be complemented for each mutation in order to perform virulence studies, and even under such conditions, issues of haplo-insufficiency add further complexity to the utility of such a multiply-marked C. albicans strain. In theory, the Ura-Blaster method overcomes this issue of limited auxotrophic markers for multiple gene disruptions by the ability to reuse the Ura3 marker. In practice however, additional problems develop, most notably the introduction of extragenic mutations which accumulate through successive counterselections on 5-FOA; which itself is a mutagenic compound. Repeated use of the procedure, for example in the construction of a double homozygote strain, may add multiple extragenic mutations; any of which can potentially contribute to phenotype(s) unlinked to either of the disrupted loci and consequently complicate interpretation of the result. Another problem common to auxotrophic mutations is the altered growth rate they impart, in addition to their potential for contributing a further variable into phenotypic analyses. For example, despite the addition of supplementary Uridine to hyphal-inducing media, CAI4 neither forms as extensive hyphae, nor switches from the budding form to hyphal form as rapidly as its Ura3+ parent strain, SC5314.
Historically auxotrophic markers have contributed tremendously to basic research of the bakers"" yeast, Saccharomyces cerevisiae. However, a clear trend towards the use of a dominant drug selectable marker has developed, principally by an international consortium of researchers participating in the S. cerevisiae genome knock out project. To this end, a single dominant selectable marker has been constructed, comprising the E. coli-derived kanamycin resistance gene, KanR, flanked by Ashbya gossypii TEF3 promoter and terminator regulatory sequence (Wach et al., 1994, Yeast 10:1793-1808; Jimenez and Davies, 1980, Nature 287:869-871). This KanMX module is expressed in S. cerevisiae and confers resistance to the Kanamycin-related aminoglycoside, geneticin, allowing selection for the desired strain when plated in the presence of the drug after transformation. The use of this KanMX module in place of auxotrophic markers solves many of the above discussed problems associated with their use. Genetic manipulations employing this dominant selectable marker can now be carried out directly in any S. cerevisiae strain. Studies comparing Kanr-marked versus wild type strains incubated together in a chemostat reveal no detectable difference in growth rate associated with the maintenance of the KanMX module. Moreover, no indirect effects on physiological, developmental, or morphological processes are detected. Because the KanMX disruption module is completely heterologous, the efficiency of proper integration into the target locus is also greatly improved, minimizing the effort to identify the correctly disrupted strain. Thus, the greatest drawback appears to be the limited number of dominant selectable markers which exist for experimental manipulation in S. cerevisiae. 
The present invention provides a novel dominant selectable marker system in fungi that is based on the nucleoside-like antibiotic, nourseothricin (NST). This compound possesses a powerful antifungal activity against C. albicans as well as S. cerevisiae. In particular, the present invention exploits the discovery of NST sensitivity in the pathogenic yeast, C. albicans, which leads to the development of a drug resistance marker useful in gene transformation and gene disruption experiments. The dominant selectable marker system of the invention facilitates: 1) gene manipulations in both clinically and experimentally relevant strains regardless of genotype and without affecting growth rate, or hyphal formation; and 2) antifungal drug discovery, including target validation and various forms of drug screening assays.
As used herein, SAT1 refers to the naturally occurring bacterial acetyltransferase genes and protein product and NAT1 refers to the naturally occurring nourseothricin N-acetyltransferase from Streptomyces noursei. Modified SAT1 and modified NAT1 refer to the modified SAT1 and NAT1 nucleic acid sequences, respectively, of the present invention used in fungus such as C. albicans. SAT and NAT refer to homologs of SAT1 and NAT1.
The present invention provides a genetically modified nourseothricin/streptothricin resistance gene derived originally from the E. coli SAT1 gene, for expression in C. albicans. Specifically, the present invention provides a nucleic acid molecule comprising (a) the nucleotide sequence of SEQ ID NO:1; or (b) a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:2 when the nucleotide sequence is translated according to the codon usage of Candida albicans. 
The present invention further provides a number of SAT expression modules, comprising promoter and terminator sequences from C. albicans genes. In one embodiment, the promoter and termination sequences are from C. albicans genes, which include but are not limited to, CaACT1, and CaPCK1.
These modules have been constructed and shown to serve as dominant nourseothricin-resistance (NSTR) gene markers for transformation of a fungal vector in C. albicans. Maintenance of the SAT expression module shows no deleterious effect on growth rate or hyphal formation. Accordingly, the present invention provides a cell, nucleic acid molecule, and vector comprising the modified SAT1 nucleic acid sequence.
The present invention also provides a SAT expression module for Polymerase Chain Reaction (PCR) based gene knock-outs which has been used to disrupt an allele of C. albicans genes CaKRE1, CaWSC4, and CaYHR036w. In addition, the present invention further provides the use of the SAT1 gene as the primary selectable marker or as a second dominant-selectable marker suitable for gene disruption in S. cerevisiae. 
The present invention provides a kit which comprises an expression vector that expresses streptothricin acetyltransferase in yeast, such as C. albicans. 
The present invention provides a strain of C. albicans that produces the SAT1 protein. The present invention provides a strain of yeast which is resistant to NST. Accordingly, the present invention provides a method of culturing yeast cells in the presence of NST, said method comprising introducing a nucleic acid molecule comprising a nucleotide sequence encoding a SAT1 protein or a nucleic acid molecule comprising a modified SAT1 in the yeast cells, and culturing the yeast cells such that SAT1 protein is expressed in the yeast cells.
The present invention further provides a method of using the modified SAT1 gene as a resistance marker for transformation and/or disruption of genes in C. albicans. 
The present invention also provides a method of using the yeast strains comprising modified SAT1 nucleotide sequence. Specifically, the present invention provides a method for introducing recombinant DNA comprising a modified SAT1 gene into C. albicans for obtaining stable transformants.
The present invention provides a method of identifying yeast cells comprising the modified SAT1 nucleic acid of the invention, which method comprises introducing the modified SAT1 nucleic acid of the invention into the yeast cells and culturing the yeast cells in the presence of nourseothricin for a time sufficient for the expression of the SAT1 protein such that yeast cells that contain the nucleic acid molecule grow faster than yeast cells that do not contain or express the nucleic acid molecule, thereby allowing the yeast cells that contain the nucleic acid molecule to be identified. The yeast cells that do not contain the nucleic acid molecule grow slowly, if at all, or they may be killed by the nourseothricin.
The present invention provides a method for enriching yeast cells comprising a first nucleic acid molecule, which method comprises introducing a mixture of the modified SAT1 nucleic acid of the invention and the first nucleic acid molecule into the yeast cells and culturing the yeast cells in the presence of nourseothricin for a time sufficient for the expression of SAT1 such that yeast cells that contain the nucleic acid molecule grow faster than yeast cells that do not contain or express the nucleic acid molecule, thereby allowing the yeast cells that contain the modified SAT1 nucleic acid molecule to be identified and recovering the yeast cells that comprise the modified SAT1 nucleic acid molecule wherein the recovered yeast cells are enriched for yeast cells that comprise the first nucleic acid molecule. The yeast cells that do not contain the nucleic acid molecule grow slowly, if at all, or they may be killed by the nourseothricin.
The present invention also provide the use of NST as a fungicide, for controlling the growth of or killing fungi, in particular pathogenic fungi such as C. albicans. NST can be used for protecting objects from contamination by such fungi.
The present invention also provides a method of inhibiting the growth of Candida albicans cells comprising contacting Candida albicans cells with a composition comprising an effective amount of nourseothricin.
The present invention also provides a method of inhibiting the growth of Saccharomyces cerevisiae cells comprising contacting Saccharomyces cerevisiae cells with a composition comprising an effective amount of nourseothricin.
The present invention also provides a method of preventing or reducing contamination of an object by a fungus comprising contacting the object with a composition comprising an effective amount of nourseothricin.
The present invention also provides a method of preventing or reducing formation on a surface of a biofilm comprising Candida albicans, said method comprising contacting the surface with a composition comprising an effective amount of nourseothricin.
The present invention provides the method of treatment of a disease in a subject caused by an infection by a pathogenic fungus which comprises administering to the subject a pharmaceutical composition comprising a pharmaceutically acceptable carrier and NST.
The present invention provides a culture medium suitable for growth of C. albicans and S. cerevisiae comprising nourseothricin.